In humans the intestine presents a barrier that prevents the absorption of plant sterols and partially blocks the absorption of cholesterol. This barrier is disrupted in the rare autosomal recessive disorder, sitosterolemia (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974)). Sitosterolemic patients Hyperabsorb the plant sterols such as sitosterol, which provide the identifying feature of this disease (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974); Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992)). These patients also hyperabsorb cholesterol and are usually hypercholesterolemic, resulting in the development of xanthomas (cholesterol deposits in skin and tendons) and premature coronary artery disease (Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992)). Unlike other forms of hyperlipidemia, sitosterolemic subjects respond to restriction in dietary cholesterol and to bile acid resin treatment with dramatic reductions in plasma cholesterol levels (Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992); T. A. Miettinen, Eur. J. Clin. Invest. 10:27 (1980); Morganroth, et al., J. Pediatr. 85:639 (1974)).
Patients with sitosterolemia have markedly elevated ( greater than 30-fold) plasma levels of plant sterols (sitosterol, stigmasterol and campesterol) as well as other neutral sterols with modified side chains (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974); Salen, et al., J. Lipid Res. 26:203 (1985); Gregg, et al., J. Clin. Invest. 77:1864 (1986)). Normal humans absorb only xcx9c5% of the 200 to 300 mg of plant sterols consumed each day (Gould, et al., Metabolism 18:652 (1969); Salen, et al., J. Clin. Invest. 49:952 (1970)). Almost all of the absorbed sitosterol is quickly secreted into the bile so that only trace amounts of sitosterol and other plant sterols remain in the blood (Gould, et al., Metabolism 18:652 (1969); Salen, et al., J. Clin. Invest. 49:952 (1970)). In contrast, subjects with sitosterolemia absorb between 15 and 60% of ingested sitosterol, and they excrete only a fraction into the bile (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974); Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992); T. A. Miettinen, Eur. J. Clin. Invest. 10:27 (1980)). The liver secretes sitosterol into the bloodstream where it is transported as a constituent of low density and high density lipoprotein particles (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974)). With the exception of the brain, the relative proportion of sterol represented by sitosterol in tissues matches that in plasma (10-25%) (Salen, et al., J. Lipid Res. 26:1126 (1985)). Hyperabsorption and inefficient secretion is not limited to plant sterols. Sitosterolemic subjects absorb a higher fraction of dietary cholesterol than normal subjects, and they secrete less cholesterol into the bile (Bhattacharyya, et al., J. Clin. Invest. 53:1033 (1974); Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver, et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992); T. A. Miettinen, Eur. J. Clin. Invest. 10, 27 (1980)). Taken together, the genetic and metabolic data indicate that sitosterolemic patients lack a gene product that normally limits the absorption and accelerates the biliary excretion of sterols (Bjorkhem and Boberg, in The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., Eds., pp. 2073 vol. 2, chap. 65 [seventh edition] (McGraw Hill, New York, 1995); Salen, et al., J. Lipid Res. 33:945 (1992)).
The molecular mechanisms that limit sterol absorption are poorly understood, but clues have emerged recently from studies of the orphan nuclear hormone receptor LXR (Repa, et al., Science 289:1524 (2000)). Mice treated with an LXR agonist have a marked decrease in cholesterol absorption and a corresponding increase in the intestinal expression of mRNA encoding the ATP binding cassette protein (ABC) 1, a membrane-associated protein that has been implicated in the transport of cholesterol (Repa, et al., Science 289:1524 (2000); Lawn, et al., J. Clin. Invest. 104:25 (1999)).
Clearly, new approaches for reducing the absorption of dietary cholesterol, for maximizing the elimination of excess cholesterol from the liver, and for treating sterol-associated disorders such as sitosterolemia would have tremendous public health benefits. The present invention addresses these and other needs.
The present invention provides nucleic acids encoding a novel ABC family sterol transporter, called ABCG8. The herein-disclosed sequences can be used for any of a number of purposes, including for the diagnosis and treatment of sterol-associated disorders, including sitosterolemia, and for the identification of molecules that associate with and/or modulate the activity of ABCG8 and, in turn, modulate the absorption of dietary cholesterol.
In one aspect, the present invention provides an isolated nucleic acid encoding an ABCG8 polypeptide, the polypeptide comprising at least about 70% amino acid sequence identity to an amino acid sequence as set forth in SEQ ID NO:4 or 8.
In one embodiment, the polypeptide specifically binds to polyclonal antibodies generated against a polypeptide that comprises an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide forms a dimer with a second ABC polypeptide, wherein the dimer comprises sterol transport activity. In another embodiment, the dimer is a heterodimer. In another embodiment, the sterol is cholesterol. In another embodiment, the second ABC polypeptide is ABCG5. In another embodiment, the ABCG5 polypeptide (1) comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence as set forth in SEQ ID NO:2 or 6; (2) selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2 or 6; (3) comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:6; (4) is encoded by a nucleic acid that hybridizes under moderately stringent (or stringent conditions) conditions to a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:1 or 5; (5) is encoded by a nucleic acid that comprises a nucleotide sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NO:1 or 5; or (6) is encoded by a nucleic acid that comprises a nucleotide sequence of SEQ ID NO:1 or 5.
In another embodiment, the nucleic acid hybridizes under moderately stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid hybridizes under stringent hybridization conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid comprises a nucleotide sequence that is at least about 70% identical to SEQ ID NO:3 or 7. In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:3 or 7. In another embodiment, the nucleic acid is greater than 500, 1000, 1500, 2000, or more nucleotides in length. In another embodiment, the nucleic acid is from a mouse or a human. In another embodiment, the nucleic acid is expressed in the intestine or the liver in the presence of an LXR agonist. In another embodiment, the nucleic acid is expressed in the liver, the jejunum, the ileum, or the duodenum.
In another aspect, the present invention provides an expression cassette comprising any of the above-described nucleic acids. In another aspect, the present invention provides an isolated cell comprising the expression cassette.
In another aspect, the present invention provides an isolated ABCG8 polypeptide, the polypeptide comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO:4 or 8.
In one embodiment, the polypeptide selectively binds to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide forms a dimer with a second ABC polypeptide, wherein the dimer comprises sterol transport activity. In another embodiment, the dimer is a heterodimer. In another embodiment, the second ABC polypeptide is ABCG5. In another embodiment, the sterol is cholesterol. In another embodiment, the polypeptide is expressed in the intestine or the liver in the presence of an LXR agonist. In another embodiment, the polypeptide is expressed in the liver, jejunum, ileum, or duodenum. In another embodiment, the polypeptide is from a mouse or a human.
In another aspect, the present invention provides antibodies generated against a polypeptide comprising an amino acid sequence having at least about 70% amino acid sequence identity to SEQ ID NO:4 or 8.
In another aspect, the present invention provides a method of making an ABCG8 polypeptide, the method comprising (i) introducing a nucleic acid encoding an ABCG8 polypeptide comprising an amino acid sequence having at least about 70% amino acid sequence identity to SEQ ID NO:4 or 8 into a host cell or cellular extract; (ii) incubating the host cell or cellular extract under conditions such that the ABCG8 polypeptide is expressed in the host cell or cellular extract.
In one embodiment, the method further comprises recovering the ABCG8 polypeptide from the host cell or cellular extract.
In another aspect, the present invention provides a method of identifying a compound useful in the treatment or prevention of a sterol-related disorder, the method comprising contacting an ABCG8 polypeptide with a test agent, and determining the functional effect of the test agent upon the polypeptide, wherein a functional effect exerted on the polypeptide by the test agent indicates that the test agent is a compound useful in the treatment or prevention of the sterol-related disorder.
In one embodiment, the sterol is cholesterol. In another embodiment, the polypeptide comprises an amino acid sequence that is at least about 70% amino acid sequence identical to an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the polypeptide is present in a cell or cell membrane. In another embodiment, the polypeptide is bound to a heterologous ABC polypeptide, forming a heterodimer. In another embodiment, the functional effect comprises an increase in the sterol transport activity of the polypeptide. In another embodiment, the functional effect comprises a physical interaction between the test agent and the polypeptide. In another embodiment, the physical interaction is detected using a direct binding assay. In another embodiment, the sterol-related disorder is sitosterolemia. In another embodiment, the sterol-related disorder is selected from the group consisting of hypercholesterolemia, hyperlipidemia, gall stones, HDL deficiency, atherosclerosis, and nutritional deficiencies.
In another aspect, the present invention provides a method of identifying a compound useful in the treatment or prevention of a sterol-related disorder, the method comprising contacting a cell with a test agent and determining the functional effect of the test agent upon the cell, wherein the cell expresses or is capable of expressing an ABCG8 polypeptide, and wherein a functional effect exerted on the cell by the test agent indicates that the test agent is a compound useful in the treatment or prevention of the sterol-related disorder.
In one embodiment, the sterol is cholesterol. In another embodiment, the polypeptide comprises an amino acid sequence that is at least about 70% amino acid sequence identical to an amino acid sequence of SEQ ID NO:4 or 8. In another embodiment, the compound produces an increase in the expression of an ABCG8 gene that encodes the polypeptide. In another embodiment, the increase in the expression of the ABCG8 gene is detected by detecting the level of ABCG8 mRNA in the cell. In another embodiment, the increase in the expression of the ABCG8 gene is detected by detecting the level of ABCG8 polypeptide in the cell. In another embodiment, the increase in the expression of the ABCG8 gene is detected by detecting the level of ABCG8 protein activity in the cell. In another embodiment, the compound modulates the level of sterol transport activity in the cell. In another embodiment, the sterol transport activity is detected by detecting the rate of sterol efflux in the cell. In another embodiment, the increase in the level of expression of the ABCG8 gene is mediated by LXR or RXR. In another embodiment, the sterol-related disorder is sitosterolemia. In another embodiment, the sterol-related disorder is selected from the group consisting of hypercholesterolemia, hyperlipidemia, gall stones, HDL deficiency, atherosclerosis, and nutritional deficiencies.
In another aspect, the present invention provides a method of treating or preventing a sterol-related disorder in a mammal, the method comprising administering to the mammal a compound that increases the level of expression or activity of an ABCG8 polypeptide in a plurality of cells of the mammal.
In one embodiment, the sterol is cholesterol. In another embodiment, the cholesterol-related disorder is sitosterolemia. In another embodiment, the sterol-related disorder is selected from the group consisting of hypercholesterolemia, hyperlipidemia, gall stones, HDL deficiency, atherosclerosis, and nutritional deficiencies. In another embodiment, the compound produces a decrease in the amount of dietary sterol that is absorbed in the mammal. In another embodiment, the compound produces a decrease in the amount of sterol that is retained in the liver of the mammal. In another embodiment, the compound inhibits the development of foam cells within the mammal. In another embodiment, the compound causes an increase in LXR or RXR activity in the mammal. In another embodiment, the compound is identified by contacting an ABCG8 polypeptide with a test agent and determining the functional effect of the test agent upon the polypeptide, wherein a functional effect exerted on the polypeptide by the test agent indicates that the test agent is a compound useful in the treatment or prevention of the sterol-related disorder. In another embodiment, the compound is identified by contacting a cell with a test agent and determining the functional effect of the test agent upon the cell, wherein the cell expresses or is capable of expressing an ABCG8 polypeptide, and wherein a functional effect exerted on the cell by the test agent indicates that the test agent is a compound useful in the treatment or prevention of the sterol-related disorder.
In another aspect, the present invention provides a method of prescreening to identify a candidate therapeutic agent that modulates ABCG8 activity in a mammal, the method comprising (i) providing a cell which comprises an ABCG8 polypeptide; (ii) providing a test compound; and (3) determining whether the amount of sterol transport activity in the cell is increased or decreased in the presence of the test compound relative to the activity in the absence of the test compound; wherein a test compound that causes an increase or decrease in the amount of sterol transport activity is a candidate therapeutic agent for modulation of ABCG8 activity in a mammal.
In one embodiment, the method further comprises a secondary step, wherein the test compound is administered to a mammal, and the absorption of dietary sterol in the mammal is detected.